KR100536983B1 - Some genes for delivery of genetic material - Google Patents

Abstract

The present invention relates to positively charged virosomes capable of effectively transporting genetic material to kill or propagate mammalian cells in vitro or in vivo. Virosome membranes include cationic and polycationic lipids, at least one viral fusion protein and at least one cell-specific marker, wherein the marker is a monoclonal antibody, antibody fragment F (ab ') for selection and binding of target cells. ₂ , Fab ', cytokines, growth factors. The present invention also provides methods for the preparation of novel virosomes and their use, in particular for use in the preparation of pharmaceutical compositions for the treatment of cancer or leukemia.

Description

Cationic virosomes for delivering genetic material

The present invention relates to novel virosomes made of positively charged liposome vesicles comprising viral glycoproteins on the membrane for effective delivery of genetic material to target sites in the fields of gene biotechnology and gene therapy. It relates to a method of making and using the same. The positively charged virosomes of the present invention are particularly suitable for specific and non-specific non-infectious transport of genes into target cells in vitro or in vivo.

Liposomes are often used as carriers for drug transport and have been widely used as protective walls to protect pharmaceuticals with short half-lives from attacks by biochemicals in body fluids. Liposomes comprising portions of the viral envelope or reconstituted membrane proteins are referred to as "virosomes" (Sizer et al., Biochemistry 26: 5106-5113, 1987). Such virosomes have been used for non-specific transport of various drugs and DNA molecules (Vainstein et al., Methods Enzymol. 101: 492-512, 1983). These virosomes fuse with the cell membrane of the target cell to deliver the material transported into the cytoplasm of the target cell in a non-controlled manner, and the unprotected material is attacked by the degradable intracellular plant.

WO 92/13525 discloses that virosomes consisting of phospholipid bilayer membranes targeted by viral spike proteins of influenza virus and cell-specific markers, such as monoclonal antibodies, have a virus-like infiltration by receptor-mediated endocytosis. It is reported that the method can effectively fuse the model membrane with animal cells. These virosomes have the disadvantage of successfully supplying chemicals and desired drugs to target sites but stably binding and transporting charge-bearing molecules such as nucleic acids with negative charges.

Recently, cell-specific transport of genetic material bound to liposomes has received increasing attention and is particularly important for use in anticancer and gene therapy. Several methods are currently used to transport DNA or RNA into cells; Viral mediated methods, lipid mediated methods, and other methods such as microinjection or electroporation. The advantages and disadvantages of currently available gene delivery techniques can be summarized as follows.

a) viral mediated gene transfer ; Retroviral vectors allow efficient and stable introduction of genes into mammalian cells. However, the efficiency of transferring genes to non-replicating cells is very low because the retrovirus is only infecting the dividing cells. In addition, using retroviruses for general stability, oncogenes can be activated. Most researchers have chosen adenoviruses with replication-binding as gene transfer vectors. Adenovirus mediated gene transfer involves inserting a desired gene into a missing adenovirus particle or forming a complex by the transferrin-polylysine bridge between the DNA to be inserted and the viral coating of the adenovirus. The disadvantage of such a highly effective system in vivo is the risk of vague infection or inflammation; Even if the E1 gene, which lacks the ability to replicate in the virus, remains, the viral genome contains a number of open reading frames that encode viral proteins (Yang et al., 1994; Proc. Natl. Acad. Sci. USA 91, 4407-4411). Expression of viral proteins by the transformed cells can induce humoral and cellular immune responses in the body of animals and humans and thus can cause inflammation and proliferation.

In the method the intermediate (Sendai virus) HVJ, foreign DNA will form the liposome complex. The liposomes are then placed on an inactivated Sendai virus . This method has already been used to deliver genes in vivo to various tissues. In addition, cell reception of antisense oligonucleotides by HVJ-liposomes has already been reported (Morishita et al., 1993; J. Cell. Biochem. 17E, 239). However, a disadvantage is that HVJ-liposomes tend to bind non-specifically to red blood cells.

b) lipid-mediated gene delivery ; Positively charged liposomes composed of cationic lipids have been shown to be easy, safe and effective for delivering genes and antisense oligonucleotides in vitro. Highly negatively charged nucleic acids spontaneously interact with positively charged liposomes. Simply mixing liposomes with a preformed positron and a polynucleotide forms a DNA-liposomal complex. However, due to the lack of fusion peptides and cell-specific markers in the liposome membranes, the efficiency of in vivo transfection is considerably low, the incubation time is long, and therefore, a large amount of drug must be administered to achieve the desired effect. As a result, unwanted side effects may occur. There is also evidence that a significant amount of cationic lipids have a toxic effect in vivo.

Small oligonucleotides are currently being tested as anti-cancer therapeutics and as anti-viral agents. Only one of the two DNA strands is transcribed to synthesize mRNA. DNA that is transcribed into RNA is called a coding strand or sense strand. Complementary, non-coding or antisense strands have the same sequence as the mRNA. When the non-coding strand is transcribed, it creates an antisense molecule capable of binding to the target (sense) mRNA. Once the antisense RNA binds to the sense RNA, the resulting double RNA molecule is not translated and the production of the protein is blocked. At all times, short antisense oligonucleotides of 18 to 22 bases effectively bind mRNA and interfere with the translation of the mRNA. By this mechanism, antisense oligonucleotides stop proliferating in human cancer cells. Genes involved in cancer exhibit their effects through overexpression of their normal structural proteins. Genes such as c-fos, c-myc, L-myc, N-myc, c-myb, abl, bcr-abl, c-raf, c-erb-2 and K-ras are potential targets for antisense cancer treatment This can be In addition, antisense oligonucleotides have the potential to replace conventional drugs, such as anti-viral agents such as antisense oligonucleotides of the tat and gag genes of human immunodeficiency virus (HIV).

Liposomal membranes composed of reconstituted viral envelopes as detailed in Stegmann et al., EMBO J. 6: 2651-2659, 1987 are referred to as virosomes. They always consist of phospholipid bilayers containing phosphatidylcholine (PC) and phosphatidylethanolamine (PE) and viral envelopes such as spike proteins stuck in the membrane. Conventional methods of binding genetic material to PC, PE-virosomes have the disadvantage of significantly lower nucleic acid binding efficiency.

Summary of the Invention

Thus, the present invention relates to novel positively charged virosomes, which are due to the specific membrane composition, resulting in long or short chain DNA or RNA, oligodeoxynucleotides, ribonucleotides, peptide nucleic acids (PNAs), ribozymes. (Enzyme-activated RNA molecule), genes, plasmids, any desired genetic material consisting of a vector can be captured very effectively to overcome the disadvantages of the prior art.

The present invention also relates to a method of reconstituting influenza virus A, in particular hemagglutinin of strain A / Singapore, into a monolayer of cationic lipid vesicles to produce an effective cationic virosome having an average diameter of about 120-180 nm. The continuous double lipid layer of virosomes is free of leaks, a disadvantage seen in the process of making many conventional virosomes. The structure of the monolayer cation bilayer directs the hydrophilic, positively charged head of the lipid toward the aqueous phase and the hydrophobic fatty acid tail towards the center of the bilayer. When viewed under an electron microscope, the reconstituted viral spike protein (hemagglutinin) binds to the lipid bilayer and extends from the surface of the cationic vesicles (FIG. 1).

The lipid composition of the vesicle membrane consists of cationic or polycation lipids and optional phospholipids such as phosphatidylethanolamine and phosphatidylcholine. In most cases, the selection of lipid membrane components based on lacquer lipids has been shown to be beneficial;

1) 100 wt% of cationic or polyvalent cationic lipids;

2) 90 to 95 wt% of cationic or polyvalent cationic lipids and 5 to 10 wt% of phosphatidylethanolamine; or

3) 45-90 wt% of cationic or polyvalent cationic lipids and 5-10 wt% of phosphatidylethanolamine; 5-50 wt% phosphatidylcholine.

In suitable embodiments, the present invention includes, but is not limited to, monoclonal antibodies, antibody fragments such as F (ab ' ₂ ) and Fab' fragments, cytokines, or growth factors useful for selective detection and binding to target cells in cationic virosomes. Non-covalently binding non-cell-specific markers. They are linked to the vesicle membrane to extend external essential activity for the detection and binding of receptors.

Binding of cell-specific markers, such as active fragments, to the preformed vesicles described in Martin et al., (J. Biol. Chem. 257: 286-288, 1982) results in non-renewable binding-which is dependent on the targeting strategy There are significant limitations. Thus, according to a suitable embodiment of the invention, the label is a preformed phosphatidylethanolamine-cross, such as N- [4- (p-maleimido = phenylbutyryl) -phosphatidylethanolamine (MPB.PE) in the presence of a surfactant Bound to a binding molecule.

For best results, the viral glycoproteins are carefully purified before reconstitution to avoid inactivation by proteolytic cleavage or reduction of intramolecular S-S bonds. Thus, conjugated markers such as the marker-MPB.PE from the unconjugated phosphatidylethanolamine-crosslinking molecule (MPB.PE) by affinity chromatography using an activated agarose matrix suitably reduced thiopropyl sepharose 6B. It is preferable to separate. A few drops of purified conjugated label (phosphatidylethanolamine-crosslinker-labeled molecular complex) are then added to a surfactant solution comprising dissolved membrane lipids, fusion peptides, and other desired mixtures of components before the cationic virosomes are formed.

Before preparing virosomes, it has proven beneficial to carry out the process of binding bivalent functional crosslinkers of phospholipids and cell-specific markers in separate processes. This process allows for better control and optimization of the surface density of the virosome membrane, particularly for the number of cell-specific markers bound to it. An improved method of controlling the concentration of protein molecules bound or embedded in the membrane is their selectivity due to the unbalanced ratio of fusion peptides (hemagglutinin) and cell-specific markers (antibody Fab 'fragments) in the virosomes. In extreme cases, by reducing or destroying the oil, the vesicles become solid or settle.

The use of antibody fragments F (ab ') ₂ and Fab' instead of using whole antibodies as cell-specific markers is particularly beneficial because such fragments are less immunogenic than whole antibodies. In addition, the absence of an Fc domain eliminates undesirable Fc-mediated biological and immunological ranges of activity, such as complementary activity and acute humoral responses through traditional pathways, thereby interacting with the antibody and its Fc-receptors in the target cell, thereby The virosome attached is removed from the surface.

Unlike known liposome compositions for nucleic acid transport, the cationic virosomes of the present invention do not need to fuse with or degrade the plasma cell membrane to enter the cytoplasm. These are 1. adsorption; 2. Entry into the host cell through a two-step mechanism of infiltration. In the first step, they bind to cell receptors, particularly glycoproteins or glycolipids of membranes with end sialic acid, via fusion peptides (hemagglutinin) or cell-specific markers, and then through receptor-mediated endocytosis Very effective combination In the case of virosomes with cell-specific markers, such as antibody fragments, these markers will further recognize the antigenic structure on the target cell surface and bind through two different binding mechanisms. Thus, the cell-specific virosomes of the present invention have cell selectivity due to endocytosis due to viral fusion peptides such as hemagglutinin as well as selectivity for various cell types due to cell-specific markers on the membrane. very big. Virosomes with Fab 'fragments that recognize antigens associated with tumors such as TAG72, CEA, 17-1A, CA19-9, or leukemias such as CD10 (CALLA = Common Acute Lympocytic Leukaemia Antigen) are described above on the cell surface. Selectively binds tumor or leukemia cells with antigen.

In the second step, virosomes are trapped in the endosome when entering the host cell through receptor-mediated endocytosis. As a result, the pH in the endosome is reduced to about 5-6 to activate the hemagglutinin fusion protein and promote the fusion of the virosome membrane with the endosome membrane. The membrane fusion reaction opens the lipid envelope of virosomes and releases the collected genetic material into the cytoplasm. Such a mechanism is considered to have significantly improved the chance of reaching the nucleus of the genetic material to be delivered before it is eliminated by thermophilic degradation or exocytosis.

1 is a micrograph of DOTAP virosomes with viral spike protein.

2 shows the pH induced fusion activity of DOTAP-Virosomes labeled with model liposomes and octadecyl rhodamine B.

Figure 3 shows the DOTAP-virosomes with FITC-OPT captured antisense bound to human small cell lung cancer cells.

4 and 5 show significant uptake and transformation efficiency of antisense-L-myc-DOTAP-virosomes into human small cell lung cancer cells.

6 shows culture of human small cell lung cancer cells different from antisense-L-myc virosomes.

7a and 7b show the transfection efficiency of pRSVcat-DOTAP virosomes against Jurkat cells.

8 shows the fusion of phospholipid-liposomes with DOTAP-virosomes. Fusion was measured by R18 test at 37 ° C.

9 shows binding of thymidine to virosome-treated NCI-H209 cells. 75 μl virosomes containing antisense, sense or msc FITC-OPT 200 picomole and 625 μl of fresh medium containing 0.5 μCi ₁₄ C-thymidine are added to 5 × 10 ⁴ cells per ml of well.

FIG. 10 shows dose-dependent inhibition of thymidine binding to NCI-H209 cells upon addition of virosomes containing antisense-L-myc-OPT. NCI-H209 cells were cultured at an initial concentration of 1 × 10 ⁵ cells per ml per well.

FIG. 11 is a culture of human small cell lung cancer cells different from 75 μl of antisense-L-myc virosome. L-myc oncogen expression was reduced in cell lines in the order of H82 <H510A <H209. The virosome of lot 1 contains less antisense-L-myc than that of lot 2.

Figure 12 shows the transduction of Sp2 cells using materials produced by two different methods.

Figure 13 shows the transduction of P3 / NS1 cells using materials produced by two different methods.

Figure 14 shows the transduction of NIH / 3T3 cells using materials produced by two different methods.

15, 16, 17 shows the growth of KG1 cells when treated with sense and antisense c-myb-DOTAP virosomes. Add 25, 50, 100 μl virosomes containing 18, 36, 72 pmol OPT. Values represent mean ± standard deviation of three experiments.

Figure 18 shows the cell growth of CEM-C3 cells when treated with DOTAP-Virosomes with the addition of 50 μl sense and antisense c-myb virosomes.

EXAMPLE

The present invention will be fully understood through the following examples. The examples are for illustrative purposes, and the present invention is not intended to be limited thereto.

Example 1

Preparation of Cationic Lipid Vesicles with Viral Hemagglutinin with Complete Fusion Activity from Influenza Viruses Captured with Antisense L-myc-FITC (= Fluorescent Labeled) -Oligodeoxynucleotide

Preparation of DOTAP Virosomes and DOTAP-Phosphatidylcholine (PC) -Virosomes

Isolation of hemagglutinin (HA) from influenza virus A / Singapore / 6/86 strains is described in Skehel and Schild (1971), Proc. Natl. Acad.Sci. See, for example, USA 79: 968-972. Briefly, the virus is grown in the dark ureter cavity of the dark turk, and purified by performing two ultracentrifugation using sucrose concentration difference. Purified virus is stabilized in a buffer comprising 7.9 mg / ml NaCl, 4.4 mg / ml trisodium citrate.2H ₂ O, 2.1 mg / ml 2-morpholinethanol sulfonic acid pH 7.3. 53 ml of a virus suspension containing 345 μl of HA per ml are pelleted using 100,000 × g ultracentrifugation for 10 minutes. 7.7 ml of a buffer surfactant solution containing 145 mM NaCl, 2.5 mM HEPES and 54 mg / ml of the non-ionic surfactant octaethylene glycol monododecylether (OEG = C ₁₂ E ₈ ) participate in the influenza virus pellet. The pellet is completely dissolved using ultrasonic grinding for 2 minutes at room temperature. The solution is ultrafiltered at 100,000 for 1 hour. The obtained supernatant contains dissolved HA trimer (1.635 mg HA / ml) and trace amounts of neuramidase. 6 mg DOTAP is added to 3.7 ml supernatant (6 mg HA) and dissolved. The solution is sterilized by passing through a 0.2 μm filter and then transferred to a glass vessel containing 1.15 g sterile microcarrier beads, suitably Biobeads SM-2. The vessel is stirred for 1 hour using a stirrer REAX2 from Heidolph (Kelheim, Germany). If necessary, this procedure is repeated up to three times using 0.58 mg Biobeads. After completing this procedure a slightly clear DOTAP virosome solution is obtained. To produce DOTAP-PC virosomes, 3 mg DOTAP and 3 mg PC were added and dissolved in the supernatant containing 6 mg HA. Subsequently the same procedure as for DOTAP virosome.

Preparation of Virosomes with Synthetic Fusion Peptides

One method of preparing cationic virosomes having synthetic fusion peptides in the membrane is as follows;

Martin et al., Irreversible coupling of Immunoglobulin fragments to preformed vesicles; J. Biol. Chem. 257: 286-288 (1982), cross-linker N- [γ-maleimidobutyryloxy] succinimide ester (GMBS in PE + GMBS → MBS-PE + N-hydroxysuccinimide reactions as detailed above) Activation of phosphatidylethanolamine (PE);

2. Preparation of activated PE and lipid vesicles (GMB-PE), where 20% phosphatidylcholine, 70% DOTAP, 10% GMB-PE is dissolved in a buffered surfactant solution as described for DOTAP virosomes containing HA . The continuous steps to create lipid vesicles are the same as those used to make DOTAP virosomes.

3. Binding of synthetic fusion peptides to lipid vesicles, wherein a peptide comprising 20 amino acids of the following amino acid sequence is used;

[SEQ ID NO 1]

The peptide also contains an N-terminus and a C-terminus. Since the C-terminal amino acid is cysteine, there is a free thiol group that can be used to bind the peptide to GMP-PE in the membrane of the lipid vesicle.

To execute the following binding reaction

PE-GMB _membrane + HS-Cys-Peptide → PE-GMB _membrane -S-Cys-Peptide

The freshly prepared lipid vesicle solution in buffer (40 mM citric acid, 35 mM sodium diphosphate, 100 mM NaCl, 2 mM EDA, pH 5.5) is mixed with the peptide solution in the same buffer. The mixture is stirred gently at 4 ° C. under nitrogen. Lipid vesicles are separated from unbound peptides by gel filtration on a High Load Superdex 200 column.

Alternatively, the fusion peptides can be bound to lipid vesicles using preformed PE-crosslinker-peptide complexes as detailed below for the Fab 'fragments of the examples.

Binding of phosphorothiate oligodeoxyribonucleotides to DOTAP virosomes

The antisense and sense oligodeoxyribonucleotide phosphorothioates (OPTs) of the L-myc gene will be used to demonstrate the high efficiency of cationic virosomes in transfection. 5'-FITC-OPTs were synthesized via phosphoramidate chemistry (Microsynth GmbH, Balgach, Switzerland). The antisense (SEQ ID NO: 2) and sense (SEQ ID NO: 3) of OPT use pentamers as follows;

[SEQ ID NO 2]

[SEQ ID NO 3]

Like antisense and sense OPTs, a mixed reference sequence (msc) consisting of nucleotides of the same length was synthesized.

1 ml DOTAP Virosome or DOTAP-PC Virosome were added next;

a) 2 mg antisense FITC-OPT (1.3 μmol);

b) 3.4 mg sense FITC-OPT (1.3 μmol); And

c) 3.1 mg msc FITC-OPT (1.3 μmol)

FITC-OPTs are dissolved and the solution is then sonicated at 26 ° C. for 2 minutes. FITC-OPTs without capture were separated from virosomes by gel filtration on a High Load Superdex 200 column (Pharmacia, Sweden). The column is balanced using sterile PBS. A void volume aliquot containing DOTAP with collected FITC-OPT is eluted with PBS and collected. The concentration of FITC-OPT collected in virosomes was determined by completely dissolving in 0.1M NaOH containing 0.1% (v / v) Triton X-100, followed by fluorescence measurement.

Binding of Fab ′ Fragments to Virosomes Using Preformed Phosphatidylethanolamine-Divalent Functional Crosslinker Molecular Complexes

Newly reduced Fab 'derived from rat monoclonal anti-CD10- (anti-CALLA) dissolved in 2.8 mL citric acid buffer (100 mM NaCl, 40 mM citric acid, 35 mM Na ₂ HPO ₄ .2H ₂ O, 2 mM EDTA, pH 5.5) 3 mg of a fragment of 0.524 mg N- [4- (p-maleimido-phenyl) -butyryl] phosphatidylethanolamine (MPB.PE) solution in 215 μl citric acid buffer containing 0.5% n-octyl-oligo-oxyethylene Add to The mixture is then incubated under nitrogen at 4 ° C. for 16 hours with gentle stirring. After incubation, unbound MPB.PE is removed in a 400 μl batch of freshly reduced wet Thiopropyl Sepharose 6B (Pharmacia, Sweden). The mixture is incubated for 4 hours at room temperature. Thiopropyl Sepharose 6B is removed by centrifugation and the resulting solution is neutralized to pH 7.0. The neutralized solution is supplemented with OEG (54 mg / ml).

The solution prepared as described above is added with a solution to prepare the DOTAP virosome. Fab'-MPB.PE molecules are inserted into the lipid bilayer during virosome formation.

Electron microscopy

Observation of the DOTAP virosomes with an electron microscope confirmed the vesicles of a suitable monolayer lamellar structure having an average diameter of 120 to 180 nm as determined by laser light scanning. The HA protein spikes of influenza virus can be clearly seen (FIG. 1).

Determining the Fusion Activity of DOTAP Virosomes

The fusion activity of the DOTAP virosomes of the present invention is described in Hoekstra et al., Biochemistry 23: 5675-5681 and Luscher et al., (1993), Arch. Virol. As described in 130: 317-326, quantitative measurements can be made based on fluorescence generation. Add a buffered OEG (C ₁₂ E ₈ ) containing DOTAP and HA to the thin film of the fluorescent probe, stir for 5 to 10 minutes to dissolve the probe, and then in the "preparation of cationic lipid vesicles" As noted, one can insert fluorescent probe octadecyl rhodamine B chloride (R18) (obtained from Molecular Probes Inc., Eugene, USA) into the DOTAP membrane at high density. Incubation of model liposomes with rhodamine-labeled DOTAP virosomes (DOTAP: liposomal lipid ratio is 1:20) shows that quenching rhodamine is diluted. Fluorescence can be measured using a Perkin-Elmer 1000 spectrophotometer at excitation and emission wavelengths of 560 nm and 590 nm, respectively. In Figure 2, the pH-induced fusion reaction of DOTAP virosomes is shown as the percentage of fluorescence (% FDQ).

Cell Receptor of Captured Antisense-L-myc-FITC-OPT

Labeling OPT with fluorescent material has proved to be very useful for studying the cell receptive mechanism of DOTAP virosomes. Human small cell lung cancer cells (ATCC-NCL-H209) expressing large amounts of L-myc gene (Nau et al., 1985; Nature 318, 69-83) were subjected to 2-well tissue culture chamber slides (Nunc, Naperville, IL). 60566, USA). 50 μl FITC-OPT-Virosome is added to each cell. They were incubated at 37 ° C. for 5, 15, 30 minutes, washed twice with PBS and then examined by fluorescence microscopy. DOTAP virosomes with captured antisense FITC-OPT bind rapidly to cells as can be seen in FIG. 3.

Examination of Biological Effects of Antisense-L-myc-FITC-OPT-DOTAP Virosomes Measured by Thymidine Binding Method

Human small cell lung cancer cells (ATCC-NCI-H209 American Type Culture Collection, Rockville, USA) were incubated in 24-well Costar plates with an initial concentration of 1 × 10 ⁵ per well, ml. After 24 hours of incubation, the medium was removed and 625 μl of fresh medium containing 0.5 μCi ¹⁴ C-thymidine ([2-14C] thymidine, 52.0 mCi / mmol; Amersham, England) and antisense, sense or msc FITC- 75 μl of DOTAP virosomes containing 0.2 nmol of OPTs are added. The culture is gently shaken with gentle stirring at 37 ° C. for 1 hour and then transferred to the incubator. After 48 hours, the cell suspension is removed, transferred to a centrifuge vial and centrifuged. The obtained cell pellet is washed twice. If the cells are not sufficiently dispersed in a single cell suspension, they are simply exposed to trypsin / EDTA solution. The cell pellet is dissolved in 1.5 ml of 0.1 M NaOH / Triton-X-100 (0.1%) solution. 3 ml liquid scintillation cocktail (Ready Protein +, Beckman, Fullerton, CA, USA) is added to 1 ml solution. ¹⁴ C-radioactive activity is counted in the liquid scintillation counter (Beckman, Fullerton, CA, USA).

4 and 5 clearly illustrate the extraordinary acceptance and transfection efficiency of antisense-L-myc-DOTAP virosomes.

In Figure 6 it is described that cells that do not express L-myc are not disturbed or affected by antisense-L-myc virosomes. In addition, empty virosomes did not show any effect on cancer cells and normal cells. Thus, in particular, anticancer treatment with antisense OPT encapsulated in the virosomes of the present invention has considerable potential since there is no aforementioned disadvantage of conventional cationic liposomes.

Example 2

Cationic lipid vesicles are made with viral hemagglutinin with full fusion activity of the influenza virus comprising the human IL-6 gene cloned at the polylinker site and the vector pcDNA3 (= pcDNA3-IL6) captured.

Preparation of DOTAP Virosomes and Binding to pcDNA3-IL-6

pcDNA3 (Invitrogen Corporation, San Diego, USA) is a 5.4kb vector designed to stably express large amounts in eukaryotic hosts. HA is isolated and purified as described in Example 1 above. 4 mg DOTAP was dissolved in 0.5 ml of a buffered surfactant solution containing 145 mM NaCl, 2.5 mM HEPES, 54 mg / ml OEG (C ₁₂ E ₈ ), pH 7.4 and then 2 ml surfactant containing 4 mg HA was added. do. To the resulting mixture is added 100 μg pcDNA3-IL-6 and dissolved. The solution is sonicated for 30 seconds. OEG is removed from Biobeads as described in Example 1.

Transduction of DOTAP Virosomes Loaded with pcDNA-IL-6 in Rat Myeloma Cells

The resulting solution consisting of DOTAP virosome loaded with pcDNA-IL-6 was diluted 1: 1000 with PBS. 20 μl and 50 μl of solution containing 1 ng and 2.5 ng pcDNA-IL-6 were added to 2 × 10 6 myeloma cells (P3 / NSI / 1-Ag4-1; Amerincan Type Culture Collection, Rockville, USA). After 48 hours of incubation, the supernatants of the cell cultures were tested for human IL-6 by ELISA. It was found to contain 20-45 pg IL-6 per ml.

Comparison of the effects of transfection of pcDNA-IL-6 loaded DOTAP liposomes and pcDNA-IL-6 virosomes

IL-6 was not found in cell culture when conventional DOTAP liposomes (without viral fusion peptide) with the same amount of pcDNA-IL-6 contained in DOTAP virosomes were transfected into myeloma cell culture. In order to achieve the same transfection results as using pcDNA-IL-6 loaded DOTAP virosomes, the amount of loaded DOTAP liposomes of pcDNA-IL-6 should be increased by about 1000 times.

Example 3

Preparation of Cationic Lipid Vesicles with Viral Hemagglutinin Trimers with Complete Fusion Activity from Influenza Viruses Including Captured pSRVcat

Prepare DOTAP virosome pRSVcat Combine

The expression vector pRSVcat (ATCC, Rockville, USA) has a CAT gene encoding chloramphenicol acetyltransferase (CAT). The enzyme catalyzes the transfer of the acetyl group of acetyl-CoA to the 3'-hydroxy position of chloramphenicol. CAT vectors are generally useful when monitoring transfection effects. pRSVcat is collected in DOTAP virosomes under the conditions described in Example 2.

Transfection of DOTAP Virosomes Loaded with pRSVcat into Jurkat Cells

Jurkat cells (106 cells / ml) are cultured with different amounts of pRSVcat-loaded DOTAP virosomes (0.0001 μl-25 μl). After 48 hours of incubation at 37 ° C., Jurkat cells are measured by CAT-ELISA assay (Boehringer Mannheim, Germany). 7A and 7B illustrate that the maximum transfection is achieved by adding 0.01 μl DOTAP virosome. In contrast to the foregoing, 0.01 μl pRSVcat-loaded DOTAP liposomes with Jurkat cells do not show any detectable CAT activity under the same conditions.

Example 4

Receive virosomes by cells

Entering virosomes into target cells is divided into two separate steps:

1. adsorption;

2. Penetration;

Adsorption is the binding of virosomes to cell receptors that are glycoproteins or glycolipids of membranes with sialic acid at their ends via HA. In the case of certain virosomes, the Fab 'fragment further recognizes the antigenic structure on the surface of the target cell and binds to the target cell by two different binding mechanisms. Thus, certain virosomes are selective for certain cell types. Virosomes with Fab 'fragments that recognize antigens associated with tumors such as TAG72, CEA, 17-1A, CA19-9 or antigens associated with leukemias such as CD10 (CALLA) and CD20 are described above on the cell surface. It can selectively bind tumor or leukemia cells with antigen. Hemagglutinin glycoproteins are carefully separated and purified. Proteolysis or reduction of intramolecular disulfide bonds (-SS-) does not inactivate them.

Infiltration is the entry of virosomes into cells by receptor-mediated endocytosis. Virosomes are captured in endosomes. The acidic pH (5-6) in the endosome promotes the fusion of the endosome membrane and the virosome membrane. Fusion is mediated by the viral spike glycoprotein hemagglutinin (HA). A membrane fusion reaction within the endosome frees the virosome from the lipid envelope and provides an inlet for providing cytosolic drug. The fusion activity of this virosome-preparation is tested by fluorescence generation. Virosomes are labeled with fluorescent probe octadecyl rhodamine B (R18) and the fusion activity of HA is monitored by fluorescence generation due to dilution of the probe from virosomes to the liposome target membrane. 8 shows the fluorescence observed when R18 labeled DOTAP-ql was added to phospholipid-liposomes. Fluorescence increases rapidly, indicating that native HA mediates fusion.

Acceptance made by cells over time

¹⁴ C-labeled virosomes were incubated with the cells to observe the cells receiving virosomes. P3 / NS1 cells were incubated with 40 μl virosomes at 37 ° C. for 5, 10, 15, 20, and 30 minutes at 1 × 10 ⁵ / mL. After washing, cells were lysed and the amount of ¹⁴ C-labeled virosomes was measured. As can be seen from Table 3, cell uptake is very fast; During the first 5 minutes, 10% of virosomes bound. Longer incubation times do not enhance acceptance. A 1 ml virosome solution comprising about 10 ¹¹ -10 ¹² virosomes, ie 4,000-40,000 virosomes per cell, binds within 5 minutes.

TABLE 3

Example 5

Strategies to use antisense to treat cancer

So-called "antisense" oligodeoxynucleotides (ODNs) are short nucleotide DNA sequences synthesized as being inversely complementary to the target nucleotide sequence of a desired mRNA. Once the RNA-DNA double helix is formed, the interpretation of the message is interrupted and the destruction of the molecule is promoted by RNase H. Delivering ODN targeting oncogen-encoded mRNA to cancer cells may in some circumstances disrupt cell proliferation and cause the cell to die.

Antisense ODN has potential as a therapeutic. As seen in many animal clinical studies and phase-I-III trials, antisense ODN for oncozin and viral genes has been shown to be active as therapeutics.

Delivery of functional DNA molecules into cells by DNA-loaded liposomes or conventional virosomes is ineffective. Thus, virosomes with positively charged lipid bilayers (cations) have been developed to deliver genetic material. Positively charged lipid bilayers react with nucleic acids and concentrate them in the vesicles formed.

Antisense-L-myc-Virosome

First found in small cell lung cancer (SCLC) cell lines, L-myc is frequently amplified and overexpressed in SCLC. 5'-FITC-phosphothioate oligodeoxyribonucleotides (OPT) were synthesized via phosphoramidite chemistry (Microsynth GmbH, Balgach, Switzerland). The following 15 monomers were used as antisense OPT (SEQ ID NO: 4) and sense OPT (SEQ ID NO: 5).

[SEQ ID NO 4]

[SEQ ID NO: 5]

Mixed sequence (msc) OPTs of the same length as antisense and sense OPT were also synthesized. Antisense OPTs covering the translational initiation site act by interfering with ribosomal translation of the target mRNA. Antisense-L-myc-phosphorthioate oligodeoxyribonucleotides are captured in virosomes. The antiproliferative effect of L-myc-antisense DNA captured in virosomes in SCLC cell lines H209, H510, H82 was evaluated. Antisense-L-myc virosomes were added to the cells of the human small cell lung cancer cell line. Sense-L-myc virosomes and msc (mixed regulatory sequences) -virosomes were used as reference (FIG. 9).

Antisense-L-myc virosomes are about 20,000 fold more active than uncaptured antisense OPT. To induce the same effect seen in FIG. 10, L-myc-antisense OPT uncaptured in micromolar range to cell culture was added to the cell culture. Cationic virosomes were more effective for ODN transport than cell capture of uncaptured ODN and were more effective than cationic liposomes.

The growth-inhibitory effect of antisense-L-myc virosomes is associated with L-myc levels expressed in three SCLC cell lines, H209, H510, H82. From FIG. 11, it was concluded that these cells that do not express the L-myc gene are not affected by antisense-L-myc virosomes. Non-cationic virosomes showed no minimal effect on normal cells and cancer cells. Because L-myc genes are frequently amplified, overexpressed in SCLC, very limited in human adult tissues, and low in expression, L-myc may be an effective target for antisense virosome therapy.

Example 6

Non-infectious delivery of plasmid based vectors for gene therapy; Transfection of Vectors to be Expressed in Mammals by Cationic Virosomes

Current attempts in cancer gene therapy are to use plasmid-based vectors that express appropriate target genes in human cancer cells ex vivo or in vivo. The following therapeutic gene targets can be evaluated; Susceptibility genes such as the Herpes simplex virus thymidine kinase (HSV-TK) gene (Moolten FL; Cancer Res. 46: 5276-5281, 1986); Genes that target the immune system to remove cancer cells such as cytokine genes (Tepper RI et al., Cell 57; 503-512, 1989); genes encoding costimulatory genes (Townsend SE et al., Science 259; 368-370, 1993); External histocompatibility genes (Plautz GE et al .; Proc. Natl. Acad. Sci. USA 90; 4645-4649, 1993); Wild type tumor suppressor gene replacement such as p53 (Chen PL et al .; Science 250; 1576-1580, 1990).

Limitations of the viral vector currently used for gene therapy, for example, lack of specificity in the tumor cells to which genes are delivered, and stability issues associated with recombination potential and secondary tumor induction potential to form a replication competence virus. Therefore, there is a need to develop a non-infectious gene delivery technique using plasmid-based expression vectors in vivo. Cationic virosomes used herein promote another non-infectious, receptor-mediated gene delivery technique.

Typical transfection efficiencies of about 5-50% using commonly available lipids. It provides virosomes that are much more efficient than commercially available liposomes, and captures DNA with virosomes to provide stable transformed cells.

The human interleukin 6 (IL-6) gene is cloned into the polylinker site of pcDNA3, where pcDNA3 is a 5.4 kb vector with very stable expression in eukaryotic hosts. The vector contains neomycin resistance markers expressed from the SV40 early promoter for selecting transformants that are stable in the presence of G418.

Vectors can be captured in three separate ways:

1. dialysis; Plasmids are collected during virosome formation. Surfactant octyl-POE (Alexis Corp., Laeufelfinger, Switzerland) is removed via dialysis.

2. biobeads; Plasmids are collected during virosome production. Surfactant OEG is removed by biobeads.

3. Ultrasonic Fracture; The plasmid is captured by DOTAP and the obtained DOTAP-liposomes are fused with DOTAP-virosomes by ultrasound.

¹⁴ C-thymidine-labeled pcDNA3-cIL-6-DNA is made to determine the zero of the collected plasmid.

Capture method Amount of plasmid collected

Dialysis (1) 0.02 μg / μl virosome

0.009 μg / μl biobead (2) virosome

Ultrasonic Disintegration (3) 0.04 µg / µl Virosome

Using virosome preparations (1)-(3), Sp2 / 0-Ag14 cells (hybrid, non-secretory, murine; ID-No; ATCC CRL-1581; 1 ml medium, cell concentration at 1 × 10 ⁵⁾ ; Referred to herein as Sp2); P3 / NSI / 1-Ag4-1 cells (non-secretory, murine; ID-No; ATCC TIB-18; referred to herein as P3 / NSI); NIH / 3T3 cells (embryo; contact inhibitory, NIH Swiss rats; ID-No; ATCC CRL-1638) were transfected. The amount of IL-6 expressed 10 after transfection is measured by ELISA (FIGS. 12, 13, 14). All cell lines are available from ATCC, 12301 Parklawn Drive, Rockville, Maryland, USA.

Transfected Sp2 and P3 / NS1 cells were selected twice by G418. After 2 months of culture, IL-6 production was measured again. The values are shown in Table 4 below.

TABLE 4

Production of IL-6 by Transfected Cells After Re-Selection with G418

Adjust the volume of lysis buffer added to the cell pellet to obtain ca.2X10 ⁶ cells per ml.

Example 7

Antisense Strategies in Leukemia Treatment

The most common hereditary abnormality in human leukemia is the Philadelphia chromosome (Ph ¹ ) translocation. The protococogen abl of chromosome 9 was transposed to a breakpoint cluster region (bcr) of chromosome 22 to form a bcr-abl hybrid gene. The abl protooncogen normally encodes a protein with enhanced tyrosine kinase activity in cells with the bcr-abl hybrid gene. Bcr-abl transcripts were found in most patients with chronic myelogenous leukemia (CML) and in patients with Ph1 acute lymphocytic leukemia. Targeting the bcr-abl gene in CML is clearly the most ideal therapeutic course. Synthetic ODN complementary to the bcr-abl transcript produced by cleaving the second or third exon of the bcr gene in contact with the second exon of c-abl inhibits Philadelphia 1 leukemia in vitro and inhibits normal bone marrow progenitor cells. Growth has been shown to be acceptable (Szczylik C et al., Science 253: 562-565, 1991). However, bcr-abl antisense treatment is limited to CML patients.

Another target molecule of antisense treatment is the myb gene. Myb is an encoding product of Protooncogen c-myb that functions as a DNA binding specific transcription factor. Suitably expressed in hematopoietic cells and required for hematopoietic cell proliferation. 18-mer antisense ODN targeted to codons 2-7 of c-myb strongly inhibited or completely eliminated clonal growth of T-cell leukemia cell line (CCRF-CEM), as well as tested primary acute myeloid Four out of five primary chronic myeloid leukemias (CMLs) in 78% of leukemias and ferns were strongly disturbed or completely eliminated (Calabretta B et al .; Proc. Natl. Acd. Sci. USA 88: 2351 -2355, 1991).

The bone marrow is removed and used as an ingredient to treat some nephropathy, including acute and chronic leukemia. Currently, leukocyte cells are removed from the bone marrow by various substances such as immunological reagents and chemotherapeutic agents. ODN-encapsulated virosomes that target one oncogen that provides growth benefits to leukemia cells are therapeutically useful and, importantly, allow for the growth of normal progenitor cells, while the normal chemistry of removing leukocytes is eliminated. More selectivity than therapies.

Antisense-c-myb virosome

Sense and antisense OPT corresponding to c-myb codons 2-9 were prepared. The sense (SEQ ID NO: 6) and antisense (SEQ ID NO: 7) c-myb sequences are as follows;

[SEQ ID NO: 6]

[SEQ ID NO: 7]

The OPT is collected in the DOTAP virosome using the same method as the L-myc DOTAP virosome. Sense and antisense c-myb virosomes are exposed to human myeloid leukemia cell line KG-1 and human acute lymphoblastic leukemia cell line CEM-C3. Proliferation of KG-1 cells depends on the protooncogen myb gene product, whereas CEM-C3 cells do not depend on the c-myb gene. KG-1 cells are cultured in 25 and 50 μl of sense and antisense c-myb virosomes containing 18, 36 and 72 pmol of sense and antisense OPT, respectively. The number of cells is measured on days 2, 3 and 4.

Adding 25 μl of sense and antisense c-myb virosomes had only marginal effect on cell growth (FIG. 15). However, addition of 50 μl (FIG. 16) and 100 μl (FIG. 17) strongly inhibits cell growth. Higher doses of sense c-myb virosomes may be used to show inhibitory effects. It can be seen that this effect is not induced by the virosome membrane, since CEM-C3 cells are not affected by the same virosome preparation (FIG. 18).

Abbreviation used in the specification

2'-OMe; 2'-O methyl

CALLA; Common Acute Lymphoblastic Leukemia Antigens

CAT; Chloramphenicol Acetyltransferase

DOTAP; N-[(1,2,3-Dioleyloxy) -propyl] -N, N, N-trimethylammoniummethyl-sulfate

FITC-OPT; N-[(1,2,3-Dioleyloxy) -propyl] -N, N, N-trimethylammonium chloride

G418; Zen tisin ^R disulfate ^(R disulfat Geneticin (G418 antibiotic))

HA; Hemagglutinin

IL-6; Interleukin 6

MPB.PE; N- [4- (p-maleimido) -phenylbutyryl] -phosphatidylethanolamine (= crosslinker-phospholipid complex)

msc; Mixed reference sequence

NA; Neuraminidase

Octyl-POE; n-octyl-oligo-

ODN; Oligodeoxynucleotides

OEG; Octaethylene glycol monododecyl ether (C ₁₂ E ₈ )

OPT; Oligodeoxyribonucleotide Phosphorothioate

PC; Phosphatidylcholine

PE; Phosphatidylethanolamine

PNA; Peptide nucleic acid

SCLC; Small cell lung cancer

SV40; Monkey virus 40

It is a primary object of the present invention to provide a positively charged lipid vesicle, wherein the vesicle consists of a cationic or polyvalent cationic lipid and an internal-normally water soluble-space, and at least one that is embedded or bound or covalently attached to the vesicle membrane. Viral fusion peptides are further included. Vesicles suitably also have at least one cell-specific marker on the membrane. It is also an object of the present invention to provide vesicles with complete biological fusion activity which basically have the same fusion activity as the native influenza virus. The fusion peptide may be a viral glycoprotein, such as hemagglutinin or a fragment thereof, or it may be a synthetic fusion peptide capable of inducing rapid fusion of endosome membranes and vesicles of a target cell after endocytosis.

The novel vesicles or virosomes are particularly useful for delivering any desired genetic material to target locations such as animal and human cells and tissues in vitro and in vivo. The novel virosomes can penetrate into proliferating cells, such as replicable cells, as well as resting cells, ie non-proliferating cells, and thus can be widely used in fields such as biotechnology, pharmaceuticals, and medicine. For use as a medicament, the virosomes of the present invention become part of the pharmaceutical composition and further include conventional additives and a pharmaceutically acceptable carrier. Suitably the pharmaceutical composition is preferably in the form of an injection, but may also be in other forms, for example emulsions, creams, gels, ointments for topical and systemic administration.

Accordingly, it is an object of the present invention to use the virosomes of the present invention in the manufacture of pharmaceutical compositions suitable for the treatment and prevention of animals or humans that can benefit from such treatment. Another object of the present invention is to use the virosome of the present invention to prepare a diagnostic kit for use in vivo and in vitro.

In one embodiment, the vesicles of the present invention are obtained by an effective hemagglutinin (HA) reconstitution process of influenza virus A. Accordingly, it is an object of the present invention to provide a method for preparing such cationic virosomes. In a suitable embodiment, the method consists in binding the genetic material to the cationic lipid vesicles. Basically, this manufacturing method consists of the following steps;

1) Non-ionic surfactants such as octaethylene glycol mono-n-dodecylether (OEG, C ₁₂ E ₈ ) to cationic lipids, viral spike glycoproteins, genetic material to be transported, optionally phosphatidylethanolamine Dissolving the resulting complex molecules, crosslinkers and cell-specific markers together; And

2) Remove the surfactants—appropriately repeatedly—with micro-carrier beads, suitably SM-2 biobead-type polystyrene beads with a mesh size of 20-50 (0.84-0.30 mm) absorbing the surfactant. Form vesicles.

In a suitable embodiment of the invention, a suitable divalent functional crosslinker is irreversibly linked to the cell-specific markers on the vesicle membrane. Cell-specific markers for the cell-receptors responsible for the selective binding of virosomes to cells are bound to the crosslinker in such a way that they have complete biological activity. It is preferred to use a crosslinker to make a preformed molecule-complexer, wherein the crosslinker is covalently bound to the cell-specific marker with phosphatidylethanolamine or phosphatidylethanolamine.

Since the virosome of the present invention is a functionally active fusion peptide, the substance captured in the cytoplasm of the target cell is released as the endosomal pH (the aforementioned criteria) is reduced. Such controlled release prolongs the retention time of the material transported into the target cell and, on the other hand, prevents the virosome from remaining undesirably long in the endosome, thereby transporting the material transported by the virosome Reduces the risk of non-specific degradation of

In another embodiment, the present invention relates to vesicles, wherein the membrane lipids consist of phosphatidylethanolamine and phosphatidylcholine, which improves specific virosome design and allows fusion peptides and cell-specific markers to be anchored to the membrane.

A "fusion peptide" refers to a peptide or protein capable of inducing and promoting a fusion action between a virosome membrane and a lipid membrane of a target cell. In most embodiments, this refers to a viral spike glycoprotein comprising a fusion peptide, in particular a fusion protein, in particular a hemagglutinin trimer of a viral surface spike, monomers thereof, or one or two truncated subunits, Viral spike glycoproteins comprising glycopeptide HA1 and HA2 comprising a functional fusion peptide. In another embodiment of the invention, the term refers to a pure fusion peptide itself or isolated from a natural source or made synthetic. In a particularly suitable embodiment of the invention, such polypeptides comprising fusion peptides refer to influenza hemagglutinin, in particular the AH ₁ N ₁ subtype. Synthetic fusion peptides are selected from the amino acid sequences shown in Table 1 below, wherein the amino acids are represented by single letter codes (see Example 6 and FIG. 2 of WO92 / 13525).

"Cross-binder" refers to an organic dysfunctional bifunctional molecule capable of binding to the surface of a vesicle prepared according to the present invention and capable of binding to a polypeptide. In a suitable embodiment of the invention, the molecule contains an N-hydroxysuccinimide group capable of binding to phosphatidylethanolamine, a maleimide group capable of conjugate to a monoclonal antibody fragment such as succinimidyl 4- (N- Maleimidomethyl) -cyclohexane-1-carboxylate, m-maleimidobenzoyl-N-hydroxysuccinimide ester, m-maleimidebenzoyl-N-hydroxy-sulfosuccinimide ester, succinimidyl 4 -(p-maleimidophenyl) -butyrate, sulfosuccinimidyl 4 (p-maleimidophenyl) butyrate; Or N-hydroxysuccinimide groups capable of binding to cytokines and photoactive azido groups, for example N-hydroxysuccinimidylsuberate (NHS-SA), N-hydroxysuccinimidyl-4 Azidobenzoate (HASAB), N-succinimidyl-6- (4'-azido-2'-nitrophenylamino) hexanoate (SANPAH), N-sulfosuccinimidyl-6- (4 '-Azido-2'-nitrophenylamino) hexanoate and the like.

TABLE 1

The crosslinking agent used for the preformed molecular complex of the crosslinking agent and the lipid is preferably using a crosslinking agent and phosphatidylethanolamine or a crosslinking agent and a cell-specific marker.

A "cell-specific" protein or label refers to a protein capable of binding to a crosslinker or crosslinker-lipid complex and also to a receptor of a target cell. In a suitable embodiment of the invention such molecules refer to cell-receptor specific compounds such as monoclonal antibodies, antibody fragments, cytokines or growth factors. Cell-specific markers provide selective sensing and binding to target cells to improve the action of fusion peptides on virosome membranes. Suitable antibody fragments consist of F (ab ' ₂ ) and Fab' fragments, and cell-specific markers include additional interleukins and other cytokines, as shown in Table 2 below.

TABLE 2

Cytokine (International Standard Abbreviation)

The term "positive lipid" as used herein refers to a so-called head-to tail amphiphile consisting of a positive component, a non-polar tail, such as N-[(1,2,3-dioleyloxy) propyl]- N, N, N-trimethylammonium chloride (DOTMA) (Felgner, er al; Proc. Natl. Acad. USA 84; 7413-7417, 1987), N-[(1,2,3-dioleyloxy-propyl ] -N, N, N-trimethylammoniummethyl-sulfate (DOTAP), Nt-butyl-N'-tetradecyl-3-tetradecylaminopropionamidine (Ruysschaert et al., Biochem, Biophys. Res. Commun. 203: 1622-1628, 1994), etc. Unless otherwise stated, the term includes polycationic lipids as defined below.

"Polycationic lipid" refers to an organic molecule comprising a polyvalent cation such as lipospermine and a nonpolar tail; 1,3-dipalmitoyl-2-phosphatidylethanolamido-spermine (DPPES) and dioctadecyl-amidoglysil spermine (DOGS) (Behr et al .; Proc. Natl. Acad. USA 86: 6982-6986, 1989); 2,3-dioleyloxy-N- [2 (sperminecarboxamide) ethyl] -N, N-dimethyl-1-propaneaminium trifluoroacetate (DOSPA); 1,3-dioleyloxy-2- (6-carboxy-spermil) -propylamide (DOSPER); N, N, N ', N'-terramethyl-N, N'-bis (2-hydroxyethyl) -2,3-dioleyloxy-1,4-butenediammonium iodide (THDOB) Etc. can be mentioned.

As used herein, "nucleic acid" or "gene material" refers to short-chain DNA or RNA, deoxyribonucleotides, oligodeoxyribonucleotides, oligodeoxyribonucleotide selenates, oligodeoxyribonucleotide phosphorothiothios. OPTs, oligodeoxyribonucleotide phosphoramidates, oligodeoxyribonucleotide methylphosphonates, peptide nucleic acids (PNAs), ribonucleotides, oligoribonucleotides, oligoribonucleotide phosphorothioates, 2'-OMe -Oligoribonucleotide phosphate, 2'-OMe-oligoribonucleotide phosphorothioate, ribozyme (RNA with enzymatic activity), gene, plasmid and vector (cloning vehicle).

"Virosome" as used herein refers to a liposomal vesicle with a bilayer membrane consisting of benign lipids, internally-suitably water-soluble-spaces, in its simplest form, the membrane further comprising viral proteins, in particular viral glycoproteins. In a suitable embodiment, the viral protein comprises at least one fusion peptide or protein having complete biological activity, in particular the spike glycoprotein hemagglutinin or neuroamidase of influenza A (A / Singapore). Viral proteins also include amino acid sequences produced by synthesis corresponding to or identical to the fusion peptides of the influenza virus described herein. Membrane lipids consist of the aforementioned cationic weaves but optionally include other natural or synthetic lipids, suitably phospholipids such as phosphatidylcholine (PC) and phosphatidylethanolamine (PE).

In many cases the cationic virosomes of the present invention are intended for in vivo or in vitro cell culture experiments, which can be successfully used without cell-specific markers on the membrane, particularly in vivo. In addition, as described herein, the membrane may further comprise at least one cell-specific marker. The average diameter of the vesicles is about 120-180 nm as measured by electron microscopy and dynamic light-dispersion.

As described herein, "complete (biological) fusion activity" refers to virosomes composed of the reconstituted viral proteins of the invention that they basically have fusion activity against the same target cells as the reconstituted native virus. Suitably the fusion of the cationic virosomes is comparable to that of native influenza virus A. Fusion activity is known in the art, especially in Hoekstra et al., Biochemistry 23: 5675-5681, 1984, or Luscher et al., Arch. Virol. The measurement is made according to the method described in 130: 317-326, 1993.

Before antisense technology can be applied therapeutically or prophylactically to patients in need of treatment, there are a number of technical issues that must be addressed in advance in the development of suitable carrier systems. For example, genetic material, such as antisense nucleotides, are unstable and must be degraded or deactivated or inactivated before they reach the target cell, so that a significant amount of material must be captured in a conventional cationic liposome. This large amount is problematic for the potential of causing toxicity in humans or animals.

Using the cationic virosomes of the present invention as carriers to deliver genetic material can overcome these problems and prevent or at least reduce side effects due to toxicity. This beneficial effect is because the cationic virosomes of the present invention can be up to 1,000 to 2,000 times more effective in transporting entrapped genetic material such as antisense oligonucleotides to target cells when compared to known liposomes or virosomes. to be. As a result, it is practically impossible to compare the performance of the cationic virosomes of the present invention with those of conventional virosomes or cationic liposomes.

Claims (29)

In a cationic lipid vesicle having a positively charged lipid bilayer membrane in which a cationic or polycationic lipid and an active viral fusion peptide are incorporated into or covalently bound to the membrane, a) The membrane comprises cations or polycationic lipids in the range of 90 to 95% by weight and phosphatidylethanolamine in the range of 5 to 10% by weight relative to the total lipids; Or cations or polycationic lipids in the range of 45-90% by weight, 5-10% by weight of phosphatidylethanolamine and 5-50% by weight of phosphatidylcholine; b) the vesicles comprise an antibody fragment Fab 'capable of selectively sensing and binding to target cells as a cell-specific marker; c) the vesicle membrane comprises a divalent functional group crosslinker attached to the membrane via a covalent bond to phosphatidylethanolamine; d) a cell specific marker is linked to the vesicle membrane via binding of a crosslinker to the free thiol group of the cell-specific marker; e) the vesicles are lipid vesicles, characterized in that the material to be transported to the target cells are collected. The method of claim 1, i) cationic lipids N-[(1,2,3-dioleyloxy) -propyl] -N, N, N-trimethylammonium chloride (DOTMA); N-[(1,2,3-dioleoloxy) -propyl] -N, N, N-trimethylammoniummethylsulfate (DOTAP) N-t-butyl-N'- tetradecyl-3- tetradecylaminopropionamidine; (ii) polycationic lipids 1,3-dipalmitoyl-2-phosphatidylethanolamidospermine (DPPES); Dioctadecyl amidoglycosyl amine (DOGS); 2,3-dioleyloxy-N- [2 (spermine-carboxamido) ethyl] -N, N-dimethyl-1-propanealuminum trifluoroacetate (DOSPA); 1,3-dioleyloxy-2- (6-carboxy-spermil) -propylamide (DOSPER); N, N, N ', N'-tetramethyl-N, N'-bis (2-hydroxyethyl) -2,3-dioleyloxy-1,4-butanediammonium iodine (THDOB) Characterized by cationic lipid vesicles. The cationic lipid vesicle of claim 1 wherein the fusion peptide is a hemagglutinin trimer. The method of claim 1, wherein the substance to be transported is a short DNA or RNA chain, deoxyribonucleotide, oligodeoxyribonucleotide, oligodeoxyribonucleotide selenate, oligodeoxyribonucleotide phosphorothioate, oligodeoxy Ribonucleotide phosphoramidate, oligodeoxyribonucleotide methylphosphonate, peptide nucleic acid, ribonucleotide, oligoribonucleotide, oligoribonucleotide phosphorothioate, 2'-OMe-oligoribonucleotide phosphate, 2'-OMe- Cationic lipid vesicles, characterized in that the material is selected from oligo ribonucleotide phosphorothioate, ribozyme, gene, plasmid, vector. The cationic lipid vesicle of claim 1 wherein the diameter of the lipid vesicle is 120-180 nm. In a method of making a lipid vesicle having a positively charged lipid bilayer membrane in which a cationic or polycationic lipid and an active virus fusion peptide are incorporated into or covalently bound to the membrane, wherein the active virus fusion protein is comprised of the vesicle membrane and the target cell membrane. Can induce and promote fusion reactions between lipids, The way to make lipid vesicles a) Purified influenza virus is dissolved in a non-ionic surfactant such as C ₁₂ E ₈ (octaethylene glycol monododecylether) or n-octyl-oligooxyethylene and hemagglutidine using ultracentrifugation Take a supernatant aliquot containing a trimer; b) creating a conjugated molecule complex consisting of a phosphatidylethanolamine (PE), a bivalent functional crosslinker, a cell-specific marker capable of selectively sensing and binding to target cells, and an unconjugated substance Wherein the cell-specific marker is an antibody fragment Fab 'and the cross-linking agent has a divalent functional group that is each bound to an amino group of PE and a thiol group of the marker; c) buffers, non-ionic surfactants; Supernatant aliquots containing hemagglutinin; Cationic or polycationic lipids; Optionally phosphatidylcholine; To make a solution (PE / crosslinker / marker molecule complex form) comprising phosphatidylethanolamine (PE), crosslinker, cell specific marker; Lipid concentrations range from 90 to 95% by weight of cationic or polycationic lipids and from 5 to 10% by weight of phosphatidylethanolamine relative to total lipids; Or to adjust to include cationic or polycationic lipids in the 45 to 90% weight range, 5 to 10% by weight phosphatidylethanolamine and 5 to 50% by weight phosphatidylcholine; d) the solution is treated with microcarrier beads, such as polystyrene Biobeads SM-2 to remove the surfactant, forming positively charged lipid bilayer vesicles; e) adding a substance to be delivered to the target cell to the vesicles, sonicating the mixture to allow the material to bind into the vesicles, and removing the unbound material through gel filtration to create the lipid vesicles. Way. 7. The method of claim 6, wherein the weight ratio of hemagglutinin based on total membrane lipids is adjusted to 1 (mg / mg) or less. 7. The method of claim 6, wherein the weight ratio of hemagglutinin to Fab 'fragment is 2: 1. The method of claim 6, i) cationic lipids N-[(1,2,3-dioleyloxy) -propyl] -N, N, N-trimethylammonium chloride (DOTMA); N-[(1,2,3-dioleoloxy) -propyl] -N, N, N-trimethylammoniummethylsulfate (DOTAP) N-t-butyl-N'- tetradecyl-3- tetradecylaminopropionamidine; (ii) polycationic lipids 1,3-dipalmitoyl-2-phosphatidylethanolamidospermine (DPPES); Dioctadecyl amidoglycosyl amine (DOGS); 2,3-dioleyloxy-N- [2 (spermine-carboxamido) ethyl] -N, N-dimethyl-1-propanealuminum trifluoroacetate (DOSPA); 1,3-dioleyloxy-2- (6-carboxy-spermil) -propylamide (DOSPER); N, N, N ', N'-tetramethyl-N, N'-bis (2-hydroxyethyl) -2,3-dioleyloxy-1,4-butanediammonium iodine (THDOB) How to feature. The method of claim 6, wherein the non-ionic surfactant is used at a concentration of 10 to 250 μmol per ml HEPES buffer. 7. The method of claim 6, wherein the microcarrier beads have a 20-50 (0.84-0.30 mm) wet mesh size and the solution is treated up to four times with the microcarrier beads. The method of claim 6, wherein the cell-specific marker is selected from monoclonal antibodies, F (ab ') ₂ , Fab'. 7. The crosslinking agent according to claim 6, wherein the crosslinking agent is a succinimidyl derivative having a divalent functional group having a maleimido group and a carboxyl group, and the derivative is selected from a bis-N-succinimidyl derivative and a photoactive succinimidyl derivative. How to. 7. The method of claim 6, wherein the unconjugated material is separated from the reaction product using gel chromatography. The unconjugated material according to claim 14, wherein the unconjugated material is unreacted N- [4- (p-maleimidophenyl) -butyryl] -phosphatidylethanolamine (MPB.PE) or 4- (N-maleimidomethyl) cyclohexane 1-carboxylate-phosphatidylethanolamine (MCC.PE). 15. The method of claim 14, wherein the gel chromatography uses affinity chromatography with reduced thiopropylsepharose 6B. 7. The material of claim 6, wherein the substance to be transported is a short DNA or RNA chain, deoxyribonucleotide, oligodeoxyribonucleotide, oligodeoxyribonucleotide selenate, oligodeoxyribonucleotide phosphorothioate, oligodeoxyribo Nucleotide phosphoramidate, oligodeoxyribonucleotide methylphosphonate, peptide nucleic acid, ribonucleotide, oligoribonucleotide, oligoribonucleotide phosphorothioate, 2'-OMe-oligoribonucleotide phosphate, 2'-OMe-oligo And a substance selected from ribonucleotide phosphorothioate, ribozyme, gene, plasmid, vector. A cationic lipid vesicle with a composition as defined in claim 1, which is made by the method according to claim 6. The cationic lipid vesicle of claim 1, which is used in a drug delivery formulation. 20. The cationic lipid vesicle of claim 19, wherein the cationic lipid vesicle is used to transport the desired genetic material to a target cell or tissue in a specific, non-invasive manner. 21. The cationic lipid vesicle of claim 20 wherein the target cell or tissue is a resting or proliferative mammalian cell. 20. The cationic lipid vesicle of claim 19 which can be used for diagnostic or medical purposes. 23. The cationic lipid vesicle of claim 22 which can be used for the prevention or treatment of diseases in humans or animals. 20. The cationic lipid vesicle of claim 19 comprising antisense oligonucleotides suitable for antisense therapies for the treatment of diseases selected from cancer, leukemia, and viral infections. 25. The cationic lipid vesicle of claim 24 wherein the antisense oligonucleotide is a protooncogene or oncogene encoded mRNA. In the method of connecting another protein to a lipid membrane containing a protein, -Linking a hetero-divalent-functional crosslinker to a lipid molecule having an available free amino group, -Connect the resulting complex (lipid / crosslinker) to the free thiol group of the protein to be bound, The complex (lipid / crosslinker / protein) is dissolved with the lipid membrane material in a solution containing a buffer, a suitable surfactant, Removing the solvent from the mixture; Removing the solvent to form a vesicle-shaped lipid membrane, wherein the membrane has a lipid / crosslinker / protein-complex bound to the other membrane. 27. The method of claim 26, wherein the surfactant is C ₁₂ E ₈ (octaethylene glycol monododecylether) or n-octyl-oligooxyethylene. The method of claim 26, wherein the protein to be bound is selected from antibodies, antibody fragments, cytokines, growth factors. 27. The method of claim 26, wherein the lipid concentration of the lipid membrane is adjusted to include 5-10 wt% phosphatidylethanolamine and 90-95 wt% phosphatidylcholine or a cationic or polycationic lipid based on total lipids.